CN101570779A - Sieving and checking method for cancers - Google Patents
Sieving and checking method for cancers Download PDFInfo
- Publication number
- CN101570779A CN101570779A CNA2008100946592A CN200810094659A CN101570779A CN 101570779 A CN101570779 A CN 101570779A CN A2008100946592 A CNA2008100946592 A CN A2008100946592A CN 200810094659 A CN200810094659 A CN 200810094659A CN 101570779 A CN101570779 A CN 101570779A
- Authority
- CN
- China
- Prior art keywords
- target gene
- chemical testing
- laboratory examination
- methylation
- corpse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention relates to a sieving and checking method for cancers, comprising the following steps: (1), providing a test body to be checked; (2), checking the CpG sequence methylation state of at least one target gene in the genome DNA of the test body to be checked, wherein at least one of the target genes comprises SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; and (3), judging whether the test body has cancers or precancerous lesions or not according to the existence or inexistence of the methylation state of the target genes. The methylation state checking method comprises a methylation specific polymerase chain reaction, a quantitative methylation specific polymerase chain reaction, sulphite sequencing, micro-arrays, mass spectrograph analysis or denaturation high-efficiency liquid chromatography, and the like.
Description
Technical field
The present invention relates to a kind of method of cancer screening, particularly a kind of with the method for methylate DNA as the cancer screening of biomarker, belong to the Medical Molecular Biology field.
Background technology
Cervical cancer is one of main cause of the death of the whole world and TaiWan, China women, and according to the statistics of The World Health Organization (WHO) in 2002, cervical cancer is second of global women's cancer cause of the death, is only second to breast cancer; Regularly accepting the cervical cancer screening is the best approach of prevention cervical cancer, and the mode of cervical cancer screening commonly used mainly contains two kinds, the one, and modal Pap test (Pap smear), another then is human mastoid process virus examination (HPV testing); Whether Pap test is the secretory product that takes out uterine cervix, in the chrotoplast, have the carninomatosis sell of one's property to give birth to, with early stage detecting cervical cancer on wherein coming off with microscopic examination; The HPV check then is with reverse transcription polymerase chain reaction (reverse transcription polymerasechain reaction, RT-PCR) check in the sample whether have human papillomavirus (human papillomavirus, HPV) performance of virogene.
Yet, because Pap test (Pap smear) needs to smear sheet by doctor's sampling, surveyor/pathology doctor interpretation, postpone the Clinics and Practices of precancerous lesion except being easy to generate high pseudo-negative rate (High false negative rate), moreover, required human cost is too high, this has the difficulty in the popularization concerning many developing countries; On the other hand, human papillomavirus's check (HPV testing) is though have high sensitive, but cause high pseudo-positive rate (High false positive rate) easily, not only allow sufferer worry in vain, also can waste many medical resources and check in the tracking of pseudo-positive patient; Therefore, how improving the accuracy and the accessibility of the cervical cancer method of inspection, is one of important topic of promoting the cervical cancer check.
On the etiology of cervical cancer, infect carcinogenic human papillomavirus (HPV) and be the most apparent risk factor; Zur Hausen shows that in report in 2002 E6/E7 carcinogenic protein (oncoprotein) meeting and caused by tumor suppressor p 53/pRB effect that " high-risk " human papillomavirus (HPV) produces cause Cycle Regulation unusual; In fact, human papillomavirus's (HPV) DNA can be detected in all cervical cancer cases.Yet, infect human papillomavirus (HPV) though, be not enough to cause the generation of cervical cancer for producing the condition of cervical cancer necessity; (the low-grade squamous intraepithelial lesions of intracutaneous pathology on the nearly 60% low squamous cell, LSIL) can restore (regress), 30% can continue (persist), 5-10% can develop into intracutaneous pathology (high-grade squamousintraepithelial lesions on the height squamous cell, HSIL), only be less than 1% and can become cervical cancer.The persistent infection of HPV and virus quantity (viral load) may be the factor of determinations that develops into intracutaneous pathology (HSIL) and cancer on the height squamous cell; Yet, the still subject to confirmation of molecular mechanism that cervical cancer takes place.
Other the factor, as: the change of environment and gene, may also in the deterioration of uterine cervix keratinocyte, play the part of important role; No matter and whether activated by HPV, the change of gene causes genomic instability to be considered to the important mechanisms that cervical cancer takes place for a long time, that is learned by cell studies show that, has nonrandom chromosomal change (non-random chromosomalchanges) in cervical cancer cell; In addition, the research of several molecular genetics then appreciation goes out some and removes heterogeneousization (loss of heterozygosity often, LOH) position, and the tumor suppressor gene when these positions may take place with cervical cancer (tumor suppressor genes, TSGs) relevant.
The disappearance of gene (genomic deletions) is considered to the important factor that tumour forms, for a long time, we have been accustomed to the coding in the genome is to be dependent on the idea that four bases of ATCG are arranged, Knudson promptly proposed the dual theory that is wound (two-hit theory) as far back as 1975, point out the sudden change that some homology tumor suppressor genes are followed or lack the generation that may cause or easily cause cancer; Yet, other message that influences phenotype (phenotype) may be stored in by in the base 5-methylcytosine (5-methylcytosine) of modified, 5-methylcytosine is found and is present among the intracellular palindromic sequence 5 ' of mammal-CpG-3 ', in the mammal cell, be called as " CpG island " (CpG islands except some, CGIs) outside the zone, most CpG dinucleotide is to all being methylated, the CpG island is meant in the zone of about 1000 base pairs (1Kb) and contains a large amount of GC-and CpG-, be usually located at gene near, and near activation of the extensive gene of performance, be found.The methylating of cytosine(Cyt) occur in DNA synthetic after, from monomethyl contributor s-gland nucleosides MET (S-adenosylmethionine, SAM) transfer to monomethyl on the position of the 5th carbon of cytosine(Cyt) through ferment, this ferment reaction system is by dnmt rna (DNA methyltransferase, DNMTs) carry out, DNMT1 is the main methyltransgerase of mammal, system is responsible for that the back is duplicated in the hemimethylation position and repairs (post-replicative restoration) for permethylated, is called as and keeps methylate (maintenance methylation); Otherwise DNMT3A and DNMT3B then are considered to mainly be responsible for the new position that methylates, and carry out a kind of step that is called again methylate (denovo methylation).
The CpG dinucleotide is to methylated loss (loss of methylation), and meaning is that general minuent methylates, and is first the super genetic abnormality (epigenetic abnormality) in the cancer cells; Yet, Nei research but shows in the past few years, (for example: (site-specific hypermethylation) is relevant with the forfeiture of its function for high methylation some tumor suppressor genes), and this may provide selective advantage (selective advantages) when cancer generates for specific position; At the high methylation that activates CpG island on the subregion, can follow silentization of the gene phenomenon (genesilencing) that continues and come by histone modification (histone modification), cause chromatin transformation (chromatin remodeling); Except chromosome deletion and transgenation, also be common in the human cancer via super silentization of the heredity phenomenon (epigenetic silencing) that activates sub tumor suppressor gene that high methylation causes.
Nearest epidemiological study shows that the concentration (a kind of main source of methyl) of Xue Qing Leaf hydrochlorate (serum folate) is with the infection of HPV and remove relevant; In the metabolism of methyl cycle (methyl cycle), the gene polytypism of ferment (genetic polymorphisms) was also once relevant with the development of intracutaneous pathology on the uterine cervix by report; As the idea that supergene develops, research related between dna methylation and cervical cancer is in vogue too, the dna methylation research of cervical cancer day with hurriedly increase, show that use methylates as the possibility of cervical cancer screening; Because the interactive characteristic of nature-nurture, the tumor suppressor gene degree that methylates is different because of different genes and different group, and different diseases also has the different phenotype that methylates (methylator phenotypes); Yet, cervical cancer methylate phenotype with and genotypic related still unknown with HPV, and have what special genes to be methylated in the cervical cancer, and need how many genes can reach the demand of clinical application, these problems are still the subject under discussion that need be identified future.
This shows that above-mentioned cervical cancer screening method commonly used still has many disappearances, demands urgently being improved.
The inventor is in view of the every shortcoming of above-mentioned cervical cancer screening method institute's deutero-commonly used,, improved innovation, and after concentrating on studies through taking great pains to attain one's goal for many years, successfully the method for cancer screening of the present invention is finished in research and development finally.
Summary of the invention
The present invention's purpose promptly is to provide a kind of method of cervical cancer screening, with the screening (cancer screen) as the first line cervical cancer.
The present invention's a time purpose is the method that is to provide a kind of cervical cancer screening, this method is except the screening that can be used as the first line cervical cancer, also can be used as the screening of the second line cervical cancer, auxiliary human papillomavirus's check (HPV testing) is to reach more accurate cervical cancer screening effect.
Another object of the present invention ties up in the method that a kind of cancer diagnosis is provided, and this method is removed and be can be applicable in the detection of cervical cancer, also can be applicable to the detection of other cancer (as: ovarian cancer, liver cancer), with the diagnosis of an auxiliary unusual corpse or other object for laboratory examination and chemical testing.
Can reach the method for a kind of cancer screening of foregoing invention purpose, be to detect the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and with the screening pointer that has or not as cancer, this method comprises the following step:
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene is made up of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; And
Wherein this tested corpse or other object for laboratory examination and chemical testing is Pap smear, ascites, blood, urine, ight soil, phlegm, oral mucosa cell, gastric juice, bile, cervical epithelial cell etc.
Wherein the CpG sequence methylation state checking method of this target gene is methylation-specific polymerase chain reaction (methylation-specific PCR, MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), sulphite sequencing (bisulfitesequencing, BS), little array (microarrays), spectrometer analysis (mass spectrometer), the sex change high performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC).
Wherein this target gene SOX1 is the nucleotide sequence that has shown in SEQ ID No:1.
Wherein this target gene PAX1 is the nucleotide sequence that has shown in SEQ ID No:2.
Wherein this target gene LMX1A is the nucleotide sequence that has shown in SEQ ID No:3.
Wherein this target gene NKX6-1 is the nucleotide sequence that has shown in SEQ ID No:4.
Wherein this target gene WT1 is the nucleotide sequence that has shown in SEQ ID No:5.
Wherein this target gene ONECUT1 is the nucleotide sequence that has shown in SEQ ID No:6.
The present invention further provides a kind of method of cervical cancer screening, is to detect the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and with the screening pointer that has or not as cervical cancer, this method comprises the following step:
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene system is made up of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; And
Wherein this tested corpse or other object for laboratory examination and chemical testing is Pap smear, blood, urine, cervical epithelial cell etc.
Wherein this tested corpse or other object for laboratory examination and chemical testing is unusual Pap smear.
Wherein this tested corpse or other object for laboratory examination and chemical testing is the be positive cervical cell corpse or other object for laboratory examination and chemical testing of (positive) of human mastoid process virus examination (HPV testing).
Wherein the CpG sequence methylation state checking method of this target gene is methylation-specific polymerase chain reaction (methylation-specific PCR, MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), sulphite sequencing (bisulfitesequencing, BS), little array (microarrays), spectrometer analysis (mass spectrometer), the sex change high performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC).
Wherein this target gene SOX1 is the nucleotide sequence that has shown in SEQ ID No:1.
Wherein this target gene PAX1 is the nucleotide sequence that has shown in SEQ ID No:2.
Wherein this target gene LMX1A is the nucleotide sequence that has shown in SEQ ID No:3.
Wherein this target gene NKX6-1 is the nucleotide sequence that has shown in SEQ ID No:4.
Wherein this target gene WT1 is the nucleotide sequence that has shown in SEQ ID No:5.
Wherein this target gene ONECUT1 is the nucleotide sequence that has shown in SEQ ID No:6.
The present invention further provides a kind of method of ovarian cancer screening, is to detect the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and with the screening pointer that has or not as ovarian cancer, this method comprises the following step:
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene system is made up of SOX1, PAX1, LMX1A; And
Wherein this tested corpse or other object for laboratory examination and chemical testing is ascites, blood, urine etc.
Wherein the CpG sequence methylation state checking method of this target gene is methylation-specific polymerase chain reaction (methylation-specific PCR, MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), sulphite sequencing (bisulfitesequencing, BS), little array (microarrays), spectrometer analysis (mass spectrometer), the sex change high performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC), tetra-sodium sequencing (pyrosequencing).
Wherein this target gene SOX1 is the nucleotide sequence that has shown in SEQ ID No:1.
Wherein this target gene PAX1 is the nucleotide sequence that has shown in SEQ ID No:2.
Wherein this target gene LMX1A is the nucleotide sequence that has shown in SEQ ID No:3.
The present invention further provides a kind of method of liver cancer screening, is to detect the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and with the screening pointer that has or not as liver cancer, this method comprises the following step:
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene is made up of SOX1, KX6-1; And
Wherein this tested corpse or other object for laboratory examination and chemical testing is ascites, blood, urine, ight soil, gastric juice, bile etc.
Wherein the CpG sequence methylation state checking method of this target gene is methylation-specific polymerase chain reaction (methylation-specific PCR, MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), sulphite sequencing (bisulfitesequencing, BS), little array (microarrays), spectrometer analysis (mass spectrometer), the sex change high performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC), tetra-sodium sequencing (pyrosequencing).
Wherein this target gene SOX1 is the nucleotide sequence that has shown in SEQ ID No:1.
Wherein this target gene NKX6-1 is the nucleotide sequence that has shown in SEQ ID No:4.
Description of drawings
See also the detailed description and the accompanying drawing thereof of following relevant a preferred embodiment of the present invention, can further understand technology contents of the present invention and purpose effect thereof; The accompanying drawing of relevant this embodiment is:
Fig. 1 has CpG sequence person to indicate with " | " for the CpG sequential analysis of employed each target gene of cancer screening method of the present invention in each gene; Each gene M SP introduction answer print fragment position indicates with "-"; Each gene sulphite sequencing (BS) introduction answer print fragment position with
Indicate;
Fig. 2 is at employed each target gene of cancer screening method of the present invention, smear in the sheet sample (10 samples mix) result who carries out methylation status of PTEN promoter (MSP) analysis at blended cervical cancer tissue sample (30 samples mix) with blended normal-sub uterine neck; The 1st hurdle is that blended normal-sub uterine neck is smeared sheet sample (10 samples mix), the 2nd hurdle is blended cervical cancer tissue sample (30 samples mixes), the negative control group in the 3rd hurdle (negative control), the positive control group in the 4th hurdle (positive control), the 5th hurdle are blank group (water);
Fig. 3 is at employed each target gene of cancer screening method of the present invention, smear in the sheet sample at discrete cervical cancer tissue sample and discrete normal-sub uterine neck, carry out the result of methylation status of PTEN promoter (MSP) analysis, T1, T2, T3, T4 represents 4 discrete cervical cancer tissue samples, N1, N2, N3, N4 represents 4 discrete normal samples originally, the field that indicates U represents to carry out with MSP introduction (U) that can the non-gene order that methylates of single-minded identification the result of methylation status of PTEN promoter (MSP), and the field that indicates M represents that the MSP introduction (M) with the gene order that can single-minded identification methylates carries out the result of methylation status of PTEN promoter (MSP);
Fig. 4 A is at employed each target gene of cancer screening method of the present invention, (AZC-in the HeLa cervical cancer cell strain of not handling 5 '-aza-2 '-deoxycytidine, 1st, 2 hurdles), and (AZC+ in the HeLa cervical cancer cell strain of handling 5 '-aza-2 '-deoxycytidine arranged, 3rd, 4 hurdles), carry out the result that methylation status of PTEN promoter (MSP) is analyzed; The field that indicates U represents to carry out with MSP introduction (U) that can the non-gene order that methylates of single-minded identification the result of methylation status of PTEN promoter (MSP), and the field that indicates M represents that the MSP introduction (M) with the gene order that can single-minded identification methylates carries out the result of methylation status of PTEN promoter (MSP);
Fig. 4 B is at employed each target gene of cancer screening method of the present invention, (AZC-in the HeLa cervical cancer cell strain of not handling 5 '-aza-2 '-deoxycytidine, the 5th hurdle), and (AZC+ in the HeLa cervical cancer cell strain of handling 5 '-aza-2 '-deoxycytidine arranged, the 6th hurdle), carry out the result of RT-PCR analysis;
Fig. 5 A is at employed each target gene of cancer screening method of the present invention, in the HeLa cervical cancer cell strain of not handling 5 '-aza-2 '-deoxycytidine, carries out the result that sulphite sequencing (BS) is analyzed;
Fig. 5 B is at employed each target gene of cancer screening method of the present invention, in the HeLa cervical cancer cell strain of handling 5 '-aza-2 '-deoxycytidine is arranged, and the result who carries out sulphite sequencing (BS) analysis;
Fig. 6 A is at employed each target gene of cancer screening method of the present invention, in uterine cervix squamous cell carcinoma (SCC), carries out the result that sulphite sequencing (BS) is analyzed;
Fig. 6 B is at employed each target gene of cancer screening method of the present invention, in normal sample, carries out the result that sulphite sequencing (BS) is analyzed.
Embodiment
Embodiment one materials and methods
One, test materials
Test materials comprises a series of complete cervix lesion samples, comprise intracutaneous pathology (LSIL on normal sample (n=45), the low squamous cell, n=45), intracutaneous pathology (HSIL on the height squamous cell, n=58), squamous cell carcinoma (squamous cell carcinoma, SCC, n=109); Test materials comprises a series of complete ovarian tumor samples in addition, comprises benign tumor of ovary sample (n=36), ovary marginality tumor sample (n=6), malignant tumor of ovary sample (n=122); All uterine cervix samples and ovary sample standard deviation are taken from the gynecological tumor tissue bank of Taibei armed forces general hospital, and the genomic dna of each sample extracts with QiageneDNA cover group, and with the quantitative DNA of PicoGreen fluorescent absorption process, and with the quality of detected through gel electrophoresis DNA.
In addition, hepatocyte samples then comprise normal liver cell sample (n=13), chronic hepatitis (n=15), liver cirrhosis (cirrhosis, n=40), liver cancer (hepatocellular carcinoma, HCC, n=54); All hepatocyte samples are all taken from Taibei armed forces general hospital general surgery tumor tissues storehouse, the genomic dna of each hepatocyte samples also is to extract with Qiagene DNA cover group, and with the quantitative DNA of PicoGreen fluorescent absorption process, and with the quality of detected through gel electrophoresis DNA.
Two, use the little array in CpG island (CpG island microarrays) carry out difference methylate the heterozygosis reaction (Differential Methylation Hybridization, DMH)
The DNA that gets 30 cervical cancer tissue's samples mixes, getting the DNA that 10 normal-sub uterine neck smear the sheet sample in addition mixes, after the DNA of sample cuts with restriction enzyme MseI enzyme, bonding (ligated) is to connexon (linkers), so that being carried out enzyme, restriction enzyme (methylation-sensitiverestriction enzymes) HpaII of the sensitivity that methylates and BstUI cut subsequently, again this DNA is carried out the amplification of 20 circulations (cycles) as the masterplate (template) of PCR, and with fluorescent stain mark, the normal-sub uterine neck is smeared the DNA of sheet sample with fluorescent stain Cy3 mark, and the DNA of cervical cancer tissue's sample is then with fluorescent stain Cy5 mark; The sample DNA that mark is good is as probe, with contain 8, the little array in CpG island of 640CpG island label (CpGisland tags) (CpG island microarrays) is carried out hybridization, with CGI database (network address: http://derlab.med.utoronto.ca/CpGIslands/) come the selected CpG island of arriving of identification.Little array data is analyzed with the circular feature pattern (circular-features mode) of GenePix 6.0 softwares, and strain (clone) is grown in the choosing that mark repeats to select, and filters and get rid of not received feature; Cy5 to the ratio (ratio) of Cy3 greater than 2.0 gene position (loci) in blended cervical cancer tissue sample, having the gene of high methylation, therefore accept ratio greater than 2.0 gene position.
Three, sulphite modification (Bisulfite modification), methylation-specific polymerase chain reaction (methylation-specific PCR, MSP) and sulphite sequencing (bisulfitesequencing, BS)
Dna modification cover group (the DNA modification kit that uses Chemicon company to produce, Chemicon, Ternecula, CA) carry out the sulphite modification: the genomic dna (genomic DNA) of getting 1 μ g sample, with S-WAT genomic dna is carried out chemically modified, in single stranded DNA, deamination all can take place and be transformed into uridylic in all non-methylated cytosine(Cyt)s, methylated cytosine(Cyt) is not then modified, and still keeps the state of 5-methylcytosine; At last, reacted sample DNA is dissolved in the TE damping fluid (TE buffer) of 55 ℃ of 70 μ l, to carry out methylation status of PTEN promoter (MSP).
Other gets the human normal DNA of blood (peripheral blood) on every side and carries out the sulphite modification, to activate the control group of subsequence as having non-methylating; And with the mankind's normal DNA (Beverly MA) handles for methyltransferase, New England Biolabs, to obtain having the positive controls of allel of methylating with the SssI methyltransgerase.
Get the sample genomic dna after 1 μ g passes through the sulphite modification, and control group and positive controls DNA, carry out the methylation status of PTEN promoter amplification with the MSP introduction, this MSP introduction is divided into two kinds, a kind ofly be MSP introduction (U) that can the non-gene order that methylates of single-minded identification, another kind of MSP introduction (M) for the gene order that can single-minded identification methylates, the MSP introduction sequence of each target gene is as shown in Table 1; The cumulative volume of methylation status of PTEN promoter reactant is 25 μ l, comprise 1 μ l masterplate DNA, each 1.5pmol of each introduction, 0.2mmol/L dNTPs and 1unit Gold Taq DNApolymerase (the Applied Biosystems of modified, Foster City, CA); With the reactant that mixes place 95 ℃ following 5 minutes, then synthesizing 30 seconds for bonding 30 seconds, 72 ℃ with 95 ℃ dissociate (denature) 30 seconds, suitable introduction bonding (annealing) temperature is circulation, dissociate, bonding, synthesis step repeats 35 circulations altogether, places 72 ℃ of reactions 5 minutes afterwards again.(ethidium bromide, 2.5% agar colloid EtBr) carries out electrophoretic analysis to product after the amplification, and places irradiation observation under the UV-light to contain ethidium bromide.
The sequence of the MSP introduction that table monomethyl specific PCR (MSP) is used
Can single-minded identification the methylate MSP introduction of gene order of introduction kind M representative.
Introduction kind U representative can the non-gene order that methylates of single-minded identification the MSP introduction.
All sample standard deviations carry out at least twice independently sulphite modification and methylation status of PTEN promoter, in the PCR reaction that the MSP introduction (M) that uses the gene order that can single-minded identification methylates is carried out, if same sample can't synthesize the PCR product more than twice, then be considered as this sample not tool methylate; The PCR product that the MSP introduction (M) that uses the gene order that can single-minded identification methylates is increased selects grows pCR4-TOPO carrier (Invitrogen, Carlsbad, CA) in, choose at least 5 independently choosing grow strain (clones) and carry out sulphite sequencing (BS), the employed introduction of sulphite sequencing (BS) as shown in Table 2, (Applied Biosystems, Foster City CA) carry out sulphite sequencing to use 377 automatic sequencing instrument.
The sequence of the introduction that table dithionite sequencing (BS) is used
Four, via 5 '-assorted nitrogen-2 '-Deoxyribose cytidine (5 '-aza-2 '-deoxycytidine) handle, make the gene performance again in the cervical cancer cell strain that methylates
At first in the strain of HeLa cervical cancer cell, may have the methylation state of the gene of high methylation with methylation status of PTEN promoter (MSP) test, and select and have methylated gene.Again dnmt rna inhibitor 5 '-aza-2 '-deoxycytidine (Sigma Chemical Co.) of HeLa cervical cancer cell strain with 10 μ M handled 4 days, make in the cell strain originally can show again because of the unexpressed gene that methylates, and with the performance of RT-PCR analyzing gene; (Qiagen, Valencia CA) extract total RNA (total RNA), and add DNase I to get rid of the pollution of DNA to use Qiagen RNeasy kit; Each sample is got 1 μ g total RNA, and (Invitrogen) to carry out cDNA with Superscript II ThermoScript II (reverse transcriptase) and 6 base random primers (random hexamer) synthetic; Synthetic cDNA carries out pcr amplification with PCR master mix reagents kit (Applied Biosystems), and place temperature cycle reactor (thermal cycler, GeneAmp 2400PE, Applied Biosystems) reaction in, with the performance of RT-PCR analyzing gene, the employed RT-PCR introduction of each target gene as shown in Table 3 again for cDNA after the amplification.
The sequence of the MSP introduction that table three RT-PCR is used
Five, human papillomavirus's (HPV) detecting
So that whether the appearance of human papillomavirus (HPV) DNA is arranged in L1consensus PCR and reverse line dot blot technology (reverse line blot) the detecting squamous cell carcinoma (SCC), if the result above this hybridization technique analyst coverage is arranged, then confirm the sequence of novel human mastoid process virus (novel HPV type) with the DNA sequencing.
Six, statistical study
Carry out data analysis with the SAS version of statistical software 9.1, gene methylate and each clinical parameter (comprising the HPV state) between relation, be to use X
2Test (X
2Test) and expense Xue Shi accurately examine and determine (Fisher ' sexact test) and calculate, and calculate and adjust stratagem ensuring success that age and HPV infect than (Odds ratios with Luo Jisi regression model (logistic regression model), ORs) and 95% confidence interval (confidenceintervals, CI), the level (the alpha level of statistical significance) that showing of statistics is ordered and is p=0.05; And calculate and use HPV and the mark that methylates (markers) to diagnose the sensitivity (sensitivity) and the specificity (specificity) of cervix lesion.
The methylate screening of pointer gene of embodiment two cervical cancers
Carry out the difference heterozygosis reaction (DMH) that methylates by the little array in CpG island (CpG island microarrays), to filter out the gene that in uterine cervix squamous cell carcinoma (SCC), has high methylation; The little array in CpG island (CpG island microarrays) result shows, cervical cancer tissue's sample and normal-sub uterine neck are smeared between the sheet sample, having 216 points has otherness and methylates, get rid of after the sequence repetition person, obtain 26 gene activation subregion CpG islands (promoter CGIs).
Carry out sequencing and analysis at these gene activation, and pick out 6 genes, these genes comprise: SOX1 (SEQ ID No:1), PAX1 (SEQ ID No:2), LMX1A (SEQ ID No:3), NKX6-1 (SEQ ID No:4), WT1 (SEQ ID No:5) and ONECUT1 (SEQ ID No:6), and its detail file are as shown in Table 4; As shown in Table 4, these six genes all are transcription factors (transcription factors) important in growth course, SOX1, PAX1, LMX1A, NKX6-1, WT1 are very important to brain, neural version (roof plate), four limbs, pancreas islet and genitourinary/urogenital growth respectively, ONECUT1 is very important to the performance of liver and pancreas gene, but the connection that studies show that these genes and cancer is seldom arranged at present.
Table four picks out the detail file of the methylated gene of tool in the cervical cancer cell with the little array in CpG island
Carry out the CpG sequential analysis at each about 500bp Nucleotide before and after above-mentioned each genetic transcription starting point (+1), as shown in Figure 1, having CpG sequence person in each gene indicates with " | ", and at its MSP introduction (as shown in Table 1) of each gene design and sulphite sequencing (BS) introduction (as shown in Table 2), each target gene carries out methylation status of PTEN promoter (MSP) and sulphite sequencing (BS) institute synthetic fragment position also is indicated among Fig. 1.
Then blended cervical cancer tissue sample (30 samples mix) is smeared sheet sample (10 samples mix) with blended normal-sub uterine neck and carry out methylation status of PTEN promoter (MSP), in different tissue samples, whether has difference with the phenomenon that methylates of confirming these 6 genes, the result as shown in Figure 2, these 6 genes are smeared the phenomenon (shown in Fig. 2 the 1st hurdle) that then do not methylate in the sheet sample all there being the phenomenon (shown in Fig. 2 the 2nd hurdle) that methylates in the blended cervical cancer tissue sample at blended normal-sub uterine neck; Further test with discrete cervical cancer tissue sample, get 4 sample (T1 of cervical cancer tissue, T2, T3, T4) and 4 normal sample (N1, N2, N3, N4) carry out methylation status of PTEN promoter (MSP), respectively with MSP introduction (U) that can the non-gene order that methylates of single-minded identification, and the MSP introduction (M) of the gene order that can single-minded identification methylates carries out methylation status of PTEN promoter (MSP), the result as shown in Figure 3, these six genes all have the phenomenon of methylating (as Fig. 3 the 1st in discrete cervical cancer tissue sample, 3,5, shown in 7 hurdles), same gene is then detected generation less than the phenomenon that methylates (as Fig. 3 the 9th in normal sample, 11,13, shown in 15 hurdles); According to The above results, with the methylate pointer gene of these 6 genes as the screening cervical cancer.
The dependency of dna methylation and gene performance in the embodiment three cervical cancer cell strains
In order to confirm that whether the methylate performance of pointer gene of cervical cancer sees through dna methylation and be used for adjusting, dnmt rna inhibitor 5 '-aza-2 '-deoxycytidine (AZC) (Sigma Chemical Co.) with 10 μ M handles HeLa cervical cancer cell strain 4 days, checks the situation of above-mentioned 6 sub-demethylations of gene activation again with methylation status of PTEN promoter (MSP); Respectively with MSP introduction (U) that can the non-gene order that methylates of single-minded identification, and the MSP introduction (M) of the gene order that can single-minded identification methylates carries out methylation status of PTEN promoter (MSP), the result is shown in Fig. 4 A, be untreated in the HeLa cervical cancer cell strain (AZC-) of 5 '-aza-2 '-deoxycytidine (AZC), 6 target genes all have the phenomenon of methylating (shown in Fig. 4 A the 1st hurdle), and detecting is less than unmethylated gene (shown in Fig. 4 A the 2nd hurdle); And in handling 4 days HeLa cervical cancer cell strain (AZC+) of 5 '-aza-2 '-deoxycytidine (AZC), then can detect unmethylated target gene (shown in Fig. 4 A the 4th hurdle), show in the cervical cancer cell strain after handling through methyltransferase inhibitors 5 '-aza-2 '-deoxycytidine (AZC) that above-mentioned 6 target genes all have part to remove to methylate.
Analyze this performance of 6 genes in the strain of HeLa cervical cancer cell with RT-PCR again, the result is shown in Fig. 4 B, in the cell strain after 5 '-aza-2 '-deoxycytidine (AZC) handles, all can detect the mRNA (shown in Fig. 4 B the 6th hurdle) of these 6 target genes, and in the cell strain without 5 '-aza-2 '-deoxycytidine (AZC) processing, then detect mRNA (shown in Fig. 4 B the 5th hurdle) less than any one target gene, by the result as can be known, these six target genes are in cervical cancer cell, really can be used for regulating its gene performance via dna methylation, when gene has the phenomenon of methylating, the performance of gene can be suppressed, remove after the methylation, target gene can begin performance again.
In the strain of HeLa cervical cancer cell, whether there is high methylation (hypermethylation) phenomenon with sulphite sequencing (BS) evaluating objects gene in addition, the result as shown in Figure 5, in the cell strain of handling without 5 '-aza-2 '-deoxycytidine (AZC) (Fig. 5 A), the sample number of target gene high methylation than the cell strain (Fig. 5 B) through 5 '-aza-2 '-deoxycytidine (AZC) processing to come many; Analyze uterine cervix squamous cell carcinoma (SCC) and normal sample with sulphite sequencing (BS) equally, the result then as shown in Figure 6, in uterine cervix squamous cell carcinoma (SCC) (Fig. 6 A) sample, it is many that the also obviously normal sample (Fig. 6 B) of the sample number of target gene high methylation comes.
The methylation analysis of embodiment four clinical uterine cervix sample internal object genes
See also table five, on normal sample, the low squamous cell on intracutaneous pathology (LSIL), the height squamous cell mean age of intracutaneous pathology (HSIL) and squamous cell carcinoma (SCC) sample be respectively 51.0 ± 11.3,39.7 ± 9.6,46.4 ± 14.4 and 53.3 ± 10.9 years old (p＜0.05); High-risk HPVDNA presents the male ratio and respectively is in the sample: normal sample 21.4%, intracutaneous pathology (LSIL) sample 47.7% on the low squamous cell, intracutaneous pathology (HSIL) sample 59.3% on the height squamous cell, squamous cell carcinoma (SCC) sample 88.9%.The result shows, the sufferer that infects HPV is more easily suffered from the cervix lesion of different severity, and (stratagem ensuring success of LSIL, HSIL, SCC sample is 3.1,5.2,29.9 than respectively; 95% confidence interval respectively is 1.1-8.3,2.1-13.0,11.5-77.7).
In the cervix lesion sample of different severity, methylation state with methylation status of PTEN promoter (MSP) evaluating objects gene, the methylation state of target gene and human papillomavirus's (HPV) the analytical results that has or not as shown in Table 5, these 6 genes of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 all have the high frequency phenomenon that methylates in squamous cell carcinoma (SCC), each gene methylated ratio in squamous cell carcinoma (SCC) sample is respectively: 81.5%, 94.4%, 89.9%, 80.4%, 77.8% and 20.4%; The ratio of each gene methylation then is respectively in normal-sub uterine neck sample: 0%, 6.7%, 11.9%, 11.1% and 0% (p≤0.001) 2.2%; Therefore, compare with normal-sub uterine neck sample, these 6 genes are obviously high by methylated situation in squamous cell carcinoma (SCC) sample.
The frequency of NKX6-1 gene methylation is 53.3% in the LSIL sample, is 55.1% in the HSIL sample, then is 80.4% in the SCC sample; Statistical result showed, the sufferer with NKX6-1 gene methylation phenomenon is suffered from the risk higher (the stratagem ensuring success ratio is that 29.8,95% confidence intervals are 10.4-85.2) of squamous cell carcinoma (SCC).
The frequency of PAX1 gene methylation is 2.3% in the LSIL sample, is 42.1% in the HSIL sample, then is 94.4% in the SCC sample; Statistical result showed has the sufferer of PAX1 gene methylation phenomenon, suffer from the height squamous cell risk of intracutaneous pathology (HSIL) and squamous cell carcinoma (SCC) higher (the stratagem ensuring success ratio of HSIL and SCC sample is＞999.9; 95% confidence interval is＜0.1-＞999.9).
Three genes of SOX1, LMX1A and ONECUT1 methylated frequency in the sample of precancerous lesion (precancerouslesions) is very low, but methylated frequency then significantly increases in HSIL sample and SCC sample, is respectively 9.3% and 81.5%, 16% and 89.9%, 7.4% and 20.4%; Statistical result showed has the sufferer of SOX1, LMX1A or ONECUT1 gene methylation phenomenon, and suffering from the risk of squamous cell carcinoma (SCC), higher (three's stratagem ensuring success ratio is respectively 200.2,124.5,7.3; 95% confidence interval is respectively 25.8-999.9,33.0-470.1,2.0-25.9).
The frequency of WT1 gene methylation increases and increases along with the pathology severity, and the frequency of WT1 gene methylation is 11.1% in normal sample, is 20.0% in the LSIL sample, is 42.1% in the HSIL sample, then is 77.8% in the SCC sample; Statistical result showed has the sufferer of WT1 gene methylation phenomenon, and suffering from the height squamous cell risk of intracutaneous pathology (HSIL) and squamous cell carcinoma (SCC), higher (both stratagem ensuring success ratios are respectively 6.7,27.9; 95% confidence interval is respectively 2.2-19.8,9.8-78.9).
The diagnosis performance of dna methylation pointer
Whether methylated sensitivity of analyzing DNA (sensitivity) and specificity (specificity) can be used as the biological pointer of height cervical cancer pathology and cervical cancer screening to determine target gene, and analytical results as shown in Table 6; Test the screening sample to have or not the sensitivity of squamous cell carcinoma (SCC) and specificity to be respectively 83.1% and 85.5% (its 95% confidence interval then is respectively 77.6-88.5 and 79.6-91.4) with HPV; And methylation state the having or not with screening squamous cell carcinoma (SCC) of analysis SOX1, PAX1, LMX1A, NKX6-1 and these 5 genes of WT1, each gene methylation state then is 77.8%-94.4% to the sensitivity of squamous cell carcinoma (SCC), and its specificity is 88.1%-100%; (combined parallel testing when merging the HPV test simultaneously and detect disease with the pointer gene that methylates individually, CPT), meaning is promptly as long as the test result of HPV test or arbitrary pointer gene that methylates is positive, the cervical cancer screening result who then assert this test sample book is positive, its sensitivity is between 97.2%-98.2%, and specificity is between 66.7%-79.5%; When merging (combined sequential testing in proper order, CST) HPV test is when methylating the pointer gene individually, meaning is promptly at first carried out the HPV test, and the sample of HPV positive test reaction is carried out the methylation state detecting of each pointer gene, its sensitivity is between 69.4%-85.0%, and the specificity of all tests is 100%.
When intracutaneous pathology (HSIL) and squamous cell carcinoma (SCC) diagnosis target, test the screening sample to have or not the sensitivity of HSIL or SCC and specificity to be respectively 75.0% and 85.5% (its 95% confidence interval then is respectively 70.2-79.8 and 79.6-91.4) on simultaneously with the height squamous cell with HPV; And the methylation state of analyzing SOX1, PAX1, LMX1A, NKX6-1 and these 5 genes of WT1 has or not HSIL or SCC with the screening sample, then between 57.4%-76.2%, its specificity is between 88.1%-100% to the sensitivity of HSIL or SCC for each gene methylation state; When merging the HPV test simultaneously and detect disease with the pointer gene that methylates individually (CPT), its sensitivity rises to 85.8%-94.9%; And when merging (CST) HPV test in proper order and methylating the pointer gene individually, the specificity of all tests is 100%; When the methylation state test of merging (CPT) HPV test and SOX1, PAX1, three genes of LMX1A simultaneously, when having or not squamous cell carcinoma (SCC) with the screening sample, its sensitivity can reach 100%, and when having or not HSIL or SCC in the screening sample in the same way, its sensitivity then is 93.4%.
In result with the pointer gene screening squamous cell carcinoma (SCC) that methylates individually, come to have or not in the screening sample sensitivity of squamous cell carcinoma (SCC) for the highest with independent detection PAX1 gene methylation state, its sensitivity can reach 94.4% (its 95% confidence interval is 90.0-98.8), same, also can reach 76.2% (its 95% confidence interval is 69.7-82.7) with the sensitivity that has or not HSIL or SCC in the PAX1 gene methylation state screening sample, the specificity of two tests then is 100%.
The methylation analysis of embodiment five ovarian tumor sample internal object genes
With the methylation state of methylation status of PTEN promoter (MSP) evaluating objects gene in the ovarian tumor sample, the methylation state analytical results of target gene as shown in Table 7, the methylation state of analysis SOX1, PAX1 and these 3 genes of LMX1A in each ovarian tumor sample, the result shows, these 3 genes of SOX1, PAX1 and LMX1A in all benign tumor of ovary and ovary marginality tumor sample, equal tool phenomenons that methylates not; And in the malignant tumor of ovary sample, the frequency of these 3 gene methylations then significantly increases, and the frequency of SOX1 gene methylation is 55.7%, and the frequency of PAX1 gene methylation is 49.2%, and the frequency of LMX1A gene methylation then is 32.8%.
The methylation state analysis of target gene in the table seven ovarian tumor sample
The methylation analysis of embodiment six hepatocyte samples internal object genes
With the methylation state of methylation status of PTEN promoter (MSP) evaluating objects gene in hepatocyte samples, the methylation state analytical results of target gene as shown in Table 8, the frequency of SOX1 gene methylation is 7.7% in the normal liver cell sample, in comparison, have in the hepatocyte samples of abnormality, the frequency of SOX1 gene methylation then significantly improves, in chronic hepatitis sample, liver cirrhosis sample and liver cancer sample, the frequency of SOX1 gene methylation is respectively 33.3%, 27.5%, 53.7%.In addition, the frequency of NKX6-1 gene methylation (10%) is also obviously low than the frequency (57%) of NKX6-1 gene methylation in the liver cancer sample in the normal liver cell sample.
The methylation state analysis of target gene in table eight hepatocyte samples
The method of cancer diagnosis provided by the present invention when comparing mutually with aforementioned located by prior art, has more following advantage:
The diagnosis pointer that the method for cancer screening provided by the present invention system has or not as cancer with the degree that methylates of specific gene in the corpse or other object for laboratory examination and chemical testing, check (HPV testing) method relatively with commonly using Pap smear and human papillomavirus, all more aforementioned both height of the susceptibility of the present invention's Method for cancer diagnostics and specificity.
The method of cancer screening provided by the present invention is except the screening that can be used as the first line cervical cancer, also can merge or auxiliary human papillomavirus check (HPV testing) check, as the screening of the second line cervical cancer, to reach more accurate cervical cancer screening effect.
The method of cancer diagnosis provided by the present invention is removed and be can be applicable in the detection of cervical cancer, also can be applicable to the detection of other cancer (as: ovarian cancer, liver cancer), with the diagnosis of an auxiliary unusual corpse or other object for laboratory examination and chemical testing.
Above-listed detailed description system specifying at one of the present invention possible embodiments, only this embodiment is not the claim in order to restriction the present invention, all do not break away from skill of the present invention spirit institute for it equivalence implement or change, for example: each target gene equivalence embodiment of variations such as judgment mode of degree that methylates in testee's corpse or other object for laboratory examination and chemical testing all should be contained in the claim of this case.
Claims (14)
1. the method for a cancer screening, this method detects the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and the screening pointer to have or not as cancer comprises the following step:
Step 1 provides a tested corpse or other object for laboratory examination and chemical testing;
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene is made up of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; And
Step 3 has or not according to this target gene methylation state, judges whether this corpse or other object for laboratory examination and chemical testing has cancer or precancerous lesion pathology.
2. the method for claim 1, it is characterized in that: a described tested corpse or other object for laboratory examination and chemical testing is Pap smear, ascites, blood, urine, ight soil, phlegm, oral mucosa cell, gastric juice, bile or cervical epithelial cell.
3. the method for claim 1 is characterized in that: the CpG sequence methylation state checking method of described target gene is methylation-specific polymerase chain reaction, quantitatively methylation-specific polymerase chain reaction, sulphite sequencing, little array, spectrometer analysis, sex change high performance liquid chromatography or tetra-sodium sequencing.
4. the method for claim 1, it is characterized in that: described target gene SOX1 is the nucleotide sequence shown in the SEQ ID No:1; Described target gene PAX1 is the nucleotide sequence shown in the SEQID No:2; Described target gene LMX1A is the nucleotide sequence shown in the SEQ ID No:3; Described target gene NKX6-1 is the nucleotide sequence shown in the SEQ ID No:4; Described target gene WT1 is the nucleotide sequence shown in the SEQ ID No:5; Described target gene ONECUT1 is the nucleotide sequence shown in the SEQ ID No:6.
5. the method for a cervical cancer screening, this method detects the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and the screening pointer to have or not as cervical cancer comprises the following step:
Step 1 provides a tested corpse or other object for laboratory examination and chemical testing;
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene is made up of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; And
Step 3 has or not according to the target gene methylation state, judges whether this corpse or other object for laboratory examination and chemical testing has cervical cancer and precancerous lesion.
6. method as claimed in claim 5 is characterized in that: a described tested corpse or other object for laboratory examination and chemical testing is Pap smear, blood, urine or cervical epithelial cell.
7. method as claimed in claim 6 is characterized in that: a described tested corpse or other object for laboratory examination and chemical testing is unusual Pap smear.
8. method as claimed in claim 6 is characterized in that: a described tested corpse or other object for laboratory examination and chemical testing is the cervical cell corpse or other object for laboratory examination and chemical testing that human mastoid process virus examination is positive.
9. the method for an ovarian cancer screening, this method detects the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, and the screening pointer to have or not as ovarian cancer comprises the following step:
Step 1 provides a tested corpse or other object for laboratory examination and chemical testing;
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene is made up of SOX1, PAX1, LMX1A; And
Step 3 has or not according to the target gene methylation state, judges whether this corpse or other object for laboratory examination and chemical testing has ovarian cancer and precancerous lesion.
10. method as claimed in claim 9 is characterized in that: a described tested corpse or other object for laboratory examination and chemical testing is ascites, blood or urine.
11. the method for a liver cancer screening, this method detect the methylated state of target gene in the tested corpse or other object for laboratory examination and chemical testing cell, the screening pointer to have or not as liver cancer comprises the following step:
Step 1 provides a tested corpse or other object for laboratory examination and chemical testing;
The CpG sequence methylation state of at least one target gene in the genomic dna of this tested corpse or other object for laboratory examination and chemical testing of step 2 detection, this target gene is made up of SOX1, NKX6-1; And
Step 3 has or not according to the target gene methylation state, judges whether this corpse or other object for laboratory examination and chemical testing has liver cancer and precancerous lesion.
12. method as claimed in claim 11 is characterized in that: a described tested corpse or other object for laboratory examination and chemical testing is ascites, blood, urine, ight soil, gastric juice or bile.
13. as claim 5,9 or 11 described methods, it is characterized in that: the CpG sequence methylation state checking method of described target gene is methylation-specific polymerase chain reaction, quantitative methylation-specific polymerase chain reaction, sulphite sequencing, little array, spectrometer analysis, sex change high performance liquid chromatography or tetra-sodium sequencing.
14. as claim 5,9 or 11 described methods, it is characterized in that: described target gene SOX1 is the nucleotide sequence shown in the SEQ ID No:1; Described target gene PAX1 is the nucleotide sequence shown in the SEQ ID No:2; Described target gene LMX1A is the nucleotide sequence shown in the SEQ ID No:3; Described target gene NKX6-1 is the nucleotide sequence shown in the SEQ ID No:4; Described target gene WT1 is the nucleotide sequence shown in the SEQ IDNo:5; Described target gene ONECUT1 is the nucleotide sequence shown in the SEQ ID No:6.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810094659.2A CN101570779B (en) | 2008-04-29 | 2008-04-29 | A kind of method of cancer screening |
| CN201610036909.1A CN105586408B (en) | 2008-04-29 | 2008-04-29 | Cancer screening method |
| HK10100677.8A HK1133283A1 (en) | 2008-04-29 | 2010-01-21 | A method for screening cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810094659.2A CN101570779B (en) | 2008-04-29 | 2008-04-29 | A kind of method of cancer screening |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610036909.1A Division CN105586408B (en) | 2008-04-29 | 2008-04-29 | Cancer screening method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101570779A true CN101570779A (en) | 2009-11-04 |
| CN101570779B CN101570779B (en) | 2016-02-17 |
Family
ID=41230264
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810094659.2A Active CN101570779B (en) | 2008-04-29 | 2008-04-29 | A kind of method of cancer screening |
| CN201610036909.1A Active CN105586408B (en) | 2008-04-29 | 2008-04-29 | Cancer screening method |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610036909.1A Active CN105586408B (en) | 2008-04-29 | 2008-04-29 | Cancer screening method |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN101570779B (en) |
| HK (1) | HK1133283A1 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974645A (en) * | 2010-11-24 | 2011-02-16 | 张秀茹 | Kit for detecting cervical carcinoma and detection method |
| CN103627784A (en) * | 2012-08-28 | 2014-03-12 | 日祥医事管理顾问股份有限公司 | Cancer screening test kit |
| CN104611410A (en) * | 2013-11-04 | 2015-05-13 | 北京贝瑞和康生物技术有限公司 | Noninvasive cancer detection method and its kit |
| CN105579590A (en) * | 2012-08-31 | 2016-05-11 | 赖鸿政 | Method for screening cancer |
| CN106367515A (en) * | 2016-09-27 | 2017-02-01 | 周宏灏 | Kit for detecting methylation degree of DNA (Deoxyribonucleic Acid) based on gold nanoparticle probe as well as detection method and application of kit |
| CN107287294A (en) * | 2017-06-14 | 2017-10-24 | 广州中心法则生物科技有限公司 | A kind of detection primer, probe, kit and its application of cervical cancer-related genes methylation |
| CN109069670A (en) * | 2016-07-29 | 2018-12-21 | 台北医学大学 | The diagnostic method of gynecological tumor |
| CN113249485A (en) * | 2021-06-24 | 2021-08-13 | 深圳市巨东生物医学工程有限公司 | Primer probe combination and kit for methylation detection of cervical cancer related genes and application of primer probe combination and kit |
| CN117721209A (en) * | 2024-02-05 | 2024-03-19 | 广州迈景基因医学科技有限公司 | Combined detection reagent and kit for cervical cancer detection |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114561462B (en) * | 2020-11-27 | 2024-01-26 | 广州达健生物科技有限公司 | Cervical cancer gene methylation detection primer probe combination, kit and application thereof |
| CN115807087A (en) * | 2022-11-04 | 2023-03-17 | 神州医疗科技股份有限公司 | Primer probe combination for methylation detection of cervical cancer PAX1-SOX1-SFRP1 gene and application thereof |
| CN117701718A (en) * | 2024-02-04 | 2024-03-15 | 湖南宏雅基因技术有限公司 | Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI335354B (en) * | 2006-09-27 | 2011-01-01 | Univ Hong Kong Chinese | Methods for the detection of the degree of the methylation of a target dna and kits |
-
2008
- 2008-04-29 CN CN200810094659.2A patent/CN101570779B/en active Active
- 2008-04-29 CN CN201610036909.1A patent/CN105586408B/en active Active
-
2010
- 2010-01-21 HK HK10100677.8A patent/HK1133283A1/en not_active IP Right Cessation
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974645A (en) * | 2010-11-24 | 2011-02-16 | 张秀茹 | Kit for detecting cervical carcinoma and detection method |
| CN103627784A (en) * | 2012-08-28 | 2014-03-12 | 日祥医事管理顾问股份有限公司 | Cancer screening test kit |
| CN113462774A (en) * | 2012-08-28 | 2021-10-01 | 湖南宏雅基因技术有限公司 | Cancer screening test kit |
| CN105579590A (en) * | 2012-08-31 | 2016-05-11 | 赖鸿政 | Method for screening cancer |
| CN104611410A (en) * | 2013-11-04 | 2015-05-13 | 北京贝瑞和康生物技术有限公司 | Noninvasive cancer detection method and its kit |
| CN109069670A (en) * | 2016-07-29 | 2018-12-21 | 台北医学大学 | The diagnostic method of gynecological tumor |
| CN109069670B (en) * | 2016-07-29 | 2022-05-10 | 北京优迅医疗器械有限公司 | Method for diagnosing gynecological tumor |
| CN106367515A (en) * | 2016-09-27 | 2017-02-01 | 周宏灏 | Kit for detecting methylation degree of DNA (Deoxyribonucleic Acid) based on gold nanoparticle probe as well as detection method and application of kit |
| CN106367515B (en) * | 2016-09-27 | 2019-12-17 | 周宏灏 | Kit for detecting DNA methylation degree based on gold nanoparticle probe and detection method and application thereof |
| CN107287294A (en) * | 2017-06-14 | 2017-10-24 | 广州中心法则生物科技有限公司 | A kind of detection primer, probe, kit and its application of cervical cancer-related genes methylation |
| CN113249485A (en) * | 2021-06-24 | 2021-08-13 | 深圳市巨东生物医学工程有限公司 | Primer probe combination and kit for methylation detection of cervical cancer related genes and application of primer probe combination and kit |
| CN117721209A (en) * | 2024-02-05 | 2024-03-19 | 广州迈景基因医学科技有限公司 | Combined detection reagent and kit for cervical cancer detection |
Also Published As
| Publication number | Publication date |
|---|---|
| HK1133283A1 (en) | 2010-03-19 |
| CN105586408A (en) | 2016-05-18 |
| CN105586408B (en) | 2021-03-23 |
| CN101570779B (en) | 2016-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101570779A (en) | Sieving and checking method for cancers | |
| Adnan et al. | Improving empirical evidence on differentiating closely related men with RM Y-STRs: a comprehensive pedigree study from Pakistan | |
| AU2004274724B2 (en) | Methods and kits useful for detecting an alteration in a locus copy number | |
| US20190136330A1 (en) | Method for screening cancer | |
| US20140193817A1 (en) | Methods and kits useful for detecting an alteration in a locus copy number | |
| WO2020020071A1 (en) | Methylation modification-based tumor marker stamp-ep1 | |
| US8980555B2 (en) | Rapid genotyping analysis and devices thereof | |
| CN109022579B (en) | Method, kit and primer set for detecting chromosome 1p/19q heterozygosity loss | |
| TR201807917T4 (en) | Methods for determining the fraction of fetal nucleic acids in maternal samples. | |
| CN107974503A (en) | Multiple lung cancer related genes methylate combined detection kit, associated detecting method and application | |
| CN109689896A (en) | Fetal chromosomal aneuploidy is detected using the region of DNA domain of the differential methylation between fetus and pregnant female animal | |
| KR20120016201A (en) | A method for screening cancer | |
| US20210024999A1 (en) | Method of identifying risk for autism | |
| EP3135768A1 (en) | Zic1 and ghsr, molecular diagnostic markers for hpv-induced invasive cancers, nonhpv-induced gynaecological and anogenital cancers and their high-grade precursor lesions | |
| KR20190059018A (en) | Method for providing information of prediction and diagnosis of obesity using methylation level of CYP2E1 gene and composition therefor | |
| CN108026583A (en) | HLA-B*15:02 single nucleotide polymorphism and its application | |
| WO2007044780A2 (en) | Methods and systems for screening for and diagnosing dna methylation associated abnormalities and sex chromosome aneuploidies | |
| CN107868822B (en) | Composition for detecting esophagus cancer, kit and application thereof | |
| WO2020221315A1 (en) | Methylation-based modified tumor marker stamp-ep8 and application thereof | |
| WO2020034888A1 (en) | Dna methylation-related marker for diagnosing tumor, and application thereof | |
| TWI385252B (en) | Cancer screening method | |
| WO2020221314A1 (en) | Tumor marker stamp-ep7 based on methylated modification and use thereof | |
| EP2450455A1 (en) | Method for determining presence or absence of epithelial cancer-origin cell in biological sample, and molecular marker and kit therefor | |
| WO2014029093A1 (en) | Method and system for determining whether individual is in abnormal state | |
| CN114787385A (en) | Methods and systems for detecting nucleic acid modifications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1133283 Country of ref document: HK |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1133283 Country of ref document: HK |